miR-122 target sites (miR-122ts)

Characteristics

Three 22-nucleotide copies, each the reverse complement of mature miR-122, joined by the 6-nucleotide spacers TACTAT and GATCAA. In the earlier AAV9 work with target arrays of 3 to 5 copies in the 3′ UTR before the BGH polyA, 3 copies reduced mouse liver luciferase 10- to 50-fold and 5 copies 20- to 100-fold in hydrodynamically injected plasmid, with no effect in 293 cells. In AAV9 (1×10^12 vg intravenous, 3 weeks), 5 copies reduced liver luciferase about 50-fold, and about 70-fold for en-MHC-TNT-LacZ. Five copies lowered transgene mRNA 96%, consistent with cleavage of a fully complementary target.

Applications: Removes leaky liver expression from intravenously delivered AAV9 vectors in mice, including the en-MHC-TNT promoter, which is strong in heart but leaky in liver. Five copies also reduced mRNA of a secretable myostatin propeptide cassette 23-fold in liver. The 3-copy array sequence is taken from a liver-detargeted cardiac genome editing vector deposited on Addgene.

Limitations: Only silences expression in miR-122-expressing hepatocytes, with no effect in tibialis anterior or 293 cells. Cardiac expression was higher with five copies (luciferase p < 0.05, LacZ p = 0.06), for an unestablished reason. The quantitative results come from 3 to 5 copy arrays in mice with plasmid and AAV9; the 3-copy array stored here was not measured in that work.

Mechanism: Each copy is fully complementary to mature miR-122, so miR-122 loaded in RISC cleaves and degrades the transgene mRNA in hepatocytes; five copies lowered a transgene mRNA 96% in mouse liver.

References

1. Qiao et al. (2010). Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver. Gene Ther - Qiao 2010 miR-122T AAV